EV71衣壳蛋白基因克隆与表达

Objective To express and purify the capsid proteins, i.e. VP1, VP2, VP3 and VP4 of EV71 in E. coli, which mainly caused hand, foot and mouth disease. Methods According to the full-length gene sequence of EV71 reported in GenBank, the primers were designed by using DNA Star software, based on which VP1-4 genes were amplified by PCR and inserted into prokaryotic expression vector pET-14b respectively. The constructed recombinant plasmid pET-14b-VPs were transformed to E. coli of BL21 strain for recombinant protein expression under induction of IPTG. The insoluble proteins were lysed in urea by supersonic, and the supernatant was collected for the purification with Ni2+-affinity chromatography, followed by SDS-PAGE. Results Restrict enzyme digestion analysis and DNA sequencing proved that recombinant plasmids pET-14b-VP1-4 were constructed correctly. We found that the VP recombinant proteins were induced, but mainly existed in the inclusion body. By means of denaturation and renaturation, we obtained VP4 protein wth high purity, while the purity of VP1-3 proteins was low and apparent molecular weights of VP1-3 proteins were not in agreement with prediction. Conclusion Prokaryotic expression plasmids for VP1-4 were successfully constructed and the recombinant proteins were induced in E. coli. Only VP4 protein was obtained with high purity, which provides a solid foundation to study the mechanism of pathogenesis of EV71.